23395953 Lecture1 Fermenter Design

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Introduction to fermentation and

Bioreactor Design

Maulik P. Suthar

Ganpat University


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Introduction

Pharmaceutical proteins produced via fermentation in transgenic microbes ormammalian cell culture systems provide economical systems for production oftherapeutic proteins. These include antibodies, vaccines, blood proteins, etc.

Biopharmaceuticals are medical drugs (see pharmacology) produced usingbiotechnology. They are proteins (including antibodies), nucleic acids (DNA,

RNA orantisense oligonucleotides) used for therapeutic orin vivo diagnosticpurposes, and are produced by means other than direct extraction from a native(non-engineered) biological source

Dozens of new pharmaceuticals produced via fermentation in transgenic microbeshave been approved for therapeutic use in the USA. Hundreds of additional biotechdrug candidates are in various stages of research or clinical trials. Fermentationsystems can be scaled up to produce quantities of pharmaceuticals that are difficult

or impossible to produce via traditional methods. Pharmaceutical quality may also beimproved. For example, pharmaceuticals produced from blood must be carefullypurified to ensure no transmission of viruses as accidental contaminants in thepharmaceutical product. Microbial systems that do not allow human viruses toreplicate enable pharmaceutical production with little or no risk of virus contamination.


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Introduction

Fermentation technology is the oldest of all biotechnological processes. Theterm is derived from the Latin verb fevere, to boil--the appearance of fruitextracts or malted grain acted upon by yeast, during the production ofalcohol.

Fermentation is a process of chemical change caused by organisms ortheir products, usually producing effervescence and heat.

Microbiologists consider fermentation as 'any process for the productionof a product by means of mass culture of micro-organisms'.

Biochemists consider fermentation as 'an energy-generating process in

which organic compounds act both as electron donors and acceptors';hence fermentation is an anaerobic process where energy is producedwithout the participation of oxygen or other inorganic electronacceptors.


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Commercially important Fermentation

Microbial cells or Biomass as the product: Eg. BakersYeast, Lactic acid bacillus, Bacillus sp.

Microbial Enzymes: Catalase, Amylase, Protease,Pectinase, Glucose isomerase, Cellulase, Hemicellulase,

Lipase, Lactase, Streptokinase etc. Microbial metabolites : Primary metabolites Ethanol, Citric acid, Glutamic acid,

Lysine, Vitamins, Polysaccharides etc. Secondary metabolites: All antibiotic fermentation

Recombinant products : Insulin, HBV, Interferon,GCSF, Streptokinase Biotransformations: Eg. Phenyl acetyl carbinol,Steroid

Biotransformation


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Metabolite

Ethanol - Saccharomycescerevisiae alcoholic beverages - Kluyveromycesfragilis Citric acid - Aspergillus nigerfood industry Acetone and Clostridium

butanol acetobutyricum solvents Lysine Corynebacterium nutritional additive Glutamic acid glutamacium flavour enhancer RiboflavinAshbyagossipiinutritional Eremothecium ashbyi Vitamin B12 Pseudomonas denitrificansnutritional

Propionibacterium shermanii Dextran Leuconostocmesenteroides industrial Xanthan gum Xanthomonascampestris industrial


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Secondary metabolites

Penicillin Penicillium chrysogenum antibiotic Erythromycin Streptomyceserythreus antibiotic Streptomycin Streptomycesgriseus antibiotic Cephalosporin Cephalosporium acrimonium antibiotic

Griseofulvin Penicillium griseofulvin antifungal antibiotic Cyclosporin A Tolypocladium inflatum immunosuppressant Gibberellin Gibberellafujikuroiplant growth regulator Secondary metabolism may be repressed in certain cases. Glucose

represses the production of actinomycin, penicillin, neomycin andstreptomycin; phosphate represses streptomycin and tetracyclinproduction. Hence, the culture medium for secondary metaboliteproduction should be carefully chosen.


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PRODUCTION ENZYMES

Aspergillus oryzae Amylases Aspergillus niger Glucamylase Trichodermareesii Cellulase

Saccharomycescerevisiea Invertase Kluyveromycesfragilis Lactase Saccharomycopsis lipolytica Lipase Aspergillus species -Pectinases and proteases

Bacillus species Proteases Mucorpusillus Microbial rennet Mucormeihei Microbial rennet


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RECOMBINANT PRODUCTS

Therapeutics

Proteins mAbs

Enzymes

Hormones DNA for gene therapy


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BIOTRANSFORMATION

Production of a structurally similar compound from aparticular one, during the fermentation process istransformation, orbiotransformation, orbioconversion. The oldest instance of this process is

the production of acetic acid from ethanol.

Immobilised plant cells may be used forbiotransformation. Using alginate as the immobilisingpolymer, digitoxin from Digitalis lanata was convertedinto digoxin, which is a therapeutic agent in greatdemand. Similarly, codeinone was converted intocodeine and tyrosine from Mucunapruriens wasconverted into DOPA.


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Vitamins

Figure 11.13, Vitamin B12

Used as supplements for human food and animal feeds

Nearly $1B/year

Synthesized chemically, but some by biocatalysis

Selected high-yield strains for B12 produce up to 60 mg/L

For riboflavin, up to 7 g/L

Amino Acids

L-Glutamate (MSG) flavor enhancer

Aspartame (phe + asp) sweetener

L-Lysine nutritive additive

DL- Methionine nutritive additive


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Design of Fermenter


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Design of Fermenter

Factors to consider when designing afermenter

Aseptic and regulatory capability, long-term reliability

Adequate aeration and agitation

Low power consumption Temperature and pH controls

Sampling facilities


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FERMENTERS AND BIOREACTORSDESIGN

There are many requirements that need to be met in the design of a large productionscale fermentation facility. Aspects of design to be considered include design yieldbasis, operating schedule, media sterilization, fermenter and ancillary vessel design,piping systems, CIP/SIP and CGMP compliance. To be successful, a well thought-outand well-designed sanitary/sterile envelope is therefore crucial to thefermentation/biotech facility.

A fermenter is the set up to carry out the process of fermentation. The fermentersvary from laboratory experimental models of one or two litres capacity, to industrialmodels of several hundred litres capacity, which refers to the volume of the mainfermenting vessel.

A bioreactordiffers from a fermenter in that the former is used for the mass cultureof plant or animal cells, instead of micro-organisms. The chemical compoundssynthesised by these cultured cells, such as therapeutic agents, can be extracted

easily from the cell biomass.

The design engineering and operational parameters of both fermenters andbioreactors are identical. With the involvement of micro-organisms as elicitors insome situations, the distinction between the two concepts is being graduallyobliterated.


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Ideal requirements of fermenter

1) Provide operation free from contamination;

2) Maintain a specific temperature;

3) Provide adequate mixing and aeration;

4) Control the pH of the culture;5) Allow monitoring and/or control of dissolved oxygen;

6) Allow feeding of nutrient solutions and reagents;

7) Provide access points for inoculation and sampling;

8) Minimize liquid loss from the vessel;

9) Facilitate the growth of a wide range of organisms


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Economy of scale


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Scale Up

Definition

Adaptation of biological methods of

production to large-scale industrial use Objectives

Obtain the best biological catalyst

Create the best possible environment Purify the products in the most economical

ways


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Typical Bioprocessing


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Three Myths of Scale-Up

We can just make it bigger.

Technology is already there.

It can be done very quickly.


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Scale-up is the key for thecommercial success

Penicillin

Process developed in England

Scaled up in the US

Commercial success by the US

High Fructose Corn Syrup

Process developed in the US Scaled up in Denmark

Commercial success by Denmark


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Penicillin Production


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Approaches for Scale-Up

Kinetics Cell Kinetics

Enzyme Kinetics

Bioreactor (Fermenter) Design Operation and Control

Separations

Engineering Aspects Transport Phenomena

Unit Operations

Cost Estimations


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Guidelines for Fermenter Design andOperation

Material: Stainless steel (Type 316) Height to diameter ratio of the vessel: 2 to 1 or 3 to 1 Impeller Two or three disk turbine impellers Diameter: 0.3 to 0.4 of tank diameter Agitation speed: 50 200 rpm Impeller shaft enters either from the top or bottom. Baffle - Four equally spaced to prevent vortex formation Width: one tenth of the tank diameter Sparger -Ring sparger (Single orifice for a small fermenter)

Heating or cooling coil For sterilization or to control the temperature


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Instrumentation and Control

The success of a fermentation process ishighly dependent on environmental factors

The fermenter needs to be able to controlsuch factors as temperature, pH, anddissolved oxygen levels


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Aeration and Agitation

Most industrial fermentations are aerobicprocesses meaning that the productionmicrobe requires oxygen to grow

The oxygen demand is met by sparging airthrough the fermentation vessel and usingan agitator increase the amount ofdissolved oxygen


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How Unit Operates

Substrate feed

Glucose, ammonia, mineral salts

Cellular metabolism of substrate Extracellular production of insulin

Air sparging for oxygen delivery

Impellers for mixing of nutrients andoxygen


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Bioreactor Specifications

HL*

= 5.5m

HL

**=

6.06m

Di1.3

m

Dt1.50

m

Total volume = 11.8 m3


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Final Design Specifications

Initial Reactor Volume 8500LMax Oxygen Demand 8mmole/gh

Target maximum kLa 2088h-1

Tank Diameter 1.5m

Impeller Diameter 1.3m

Tank Height (HL) 4.81mHL* 5.51m

HL** 6.06m

Rpm 45

Qg 0.036m3/s

Ni 2

Corrected Power (Pg) 6789W

Pg/V 776.21W/m3

Design kLa 1998h-1

Pm 8.06W/kg

Geometry Correction

Factor ()

0.13


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Companies produce Insulin

Sanofi Pasteur

Novonordisk

Eli Lilly Average cost = $40/100 I.U. or ~$800/kg

Final production cost ~$12/kg precursor

Includes capital and operating costs (minusmedium)


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Novonordisk Bioreactor


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Steps

1. Organism selection, with regard to:

substrate versatility

byproduct formation characteristics robustness of the organism, e.g.,to processupsets

viability with regard to cell recycling

physiological characteristics (maximumgrowth rate, aeration requirements, etc.)

genetic accessibility


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2. Metabolic and cellular engineering:

improve existing properties of the

organism introduce novel functions, forexample, by simplify- ing product recovery,expanding substrate and product ranges,and enabling fermentation to occur under

nonstandard conditions


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3. Fermentation process development:

culture and media optimization (fromcomplex to defined minimal media)

optimization of cultivation parameters thattake into account product recovery andpurification (minimize byproduct formation,

minimize chemical inputs, and develophigh-cell-density cultivation)

incorporation of cell retention/recycling


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Roadmap for Integrated ProcessDevelopment

Analyze of economic and process constraints based on preliminary processdesign

Identify opportunities for improvement, e.g., reduced waste streams, energyuse, impurity levels and raw material use

Put together a wish list of physiological characteristics and downstreamseparation performance

Evaluate feasibility of achieving the wish list based on technical difficultyand economics

Define the best strategy for addressing each opportunity by taking intoaccount both downstream and fermentation capabilities, such as high celldensity, extractive fermentation, simplify broth, etc.

Integrated fermentation and downstream process development


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Types of Fermenter

1. Activated sludge Fermenter2. Air Lift Fermenter3. Bubble cap Fermenter

4. Loop Fermenter5. Mist Fermenter6. Packed Bed Fermenter7. Rotating Drug Fermenter

8. Tower Fermenter9. Trickling Film Fermenter


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Classification of Fermenters


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1. Activated sludge Fermenter


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2. Air-lift Fermenters


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2. Air-lift Fermenters


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3. Bubble cap Fermenter


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4. Loop Fermenter


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5. Mist Fermenter


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6. Packed Bed Fermenter


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7. Rotating Drum Fermenter


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8. Tower/Column Fermenters


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9. Trickling Film Fermenter


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References

Stanbury, P.F., A. Whitaker, and S. J. Hall,Principles of Fermentation Technology, 2nd

ed., Butterworth Heinemann, Oxford,2000.


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References


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References


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References

23395953 Lecture1 Fermenter Design

Documents

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